The disclosed invention is generally in the field of molecular cloning and nucleic acid amplification, and specifically involves rolling circle replication of nucleic acid molecules inserted into circular vectors.
DNA molecular cloning is routinely carried out using plasmid, phage, or viral vectors that replicate inside cells. A method, in which individual DNA molecules are cloned in solution by serial dilution and subsequent PCR amplification from tubes containing single molecules has been described (Lukyanov et al., Nucleic Acid Research 24:2194-2195 (1996)). A method has also been described for cloning RNA populations derived from single RNA molecules in an immobilized medium (Chetverina and Chetverin, Nucleic Acids Research 21:2349-2353 (1993)). While both of these methods allow in vitro cloning, neither is practical for high throughput cloning.
Velculescu et al., Science 270:484-487 (1995), have described a method for the quantitative cataloguing and comparison of expressed genes in normal, developmental, and disease states. The method, termed serial analysis of gene expression (SAGE), is based in the use of relatively short sequence tags for the unique identification of cDNAs derived from mRNA transcripts. While this method is very powerful, the study of low-abundance mRNAs can require several months of work in order to obtain sufficient sequence information for a complete SAGE analysis of one tissue sample. Thus, there is a need for a method to obtain the sequence of sequence tags more rapidly.
It is therefore an object of the present invention to provide a more efficient method of in vitro molecular cloning.
It is also an object of the present invention to provide vectors and kits useful for in vitro cloning.
It is also an object of the present invention to provide an automated method molecular cloning.
It is also an object of the present invention to provide a more efficient method of sequential analysis of gene expression.